JP6709024B2 - ペプチド又はタンパク質の分画方法 - Google Patents
ペプチド又はタンパク質の分画方法 Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims description 110
- 238000000034 method Methods 0.000 title claims description 99
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- 102000004196 processed proteins & peptides Human genes 0.000 claims description 60
- 238000000926 separation method Methods 0.000 claims description 57
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 46
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- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
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- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
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- 238000001179 sorption measurement Methods 0.000 claims description 4
- LRMSQVBRUNSOJL-UHFFFAOYSA-N 2,2,3,3,3-pentafluoropropanoic acid Chemical compound OC(=O)C(F)(F)C(F)(F)F LRMSQVBRUNSOJL-UHFFFAOYSA-N 0.000 claims description 3
- IPFHQCGGKGZQFJ-UHFFFAOYSA-N 2,2,3,3,4-pentafluorobutanoic acid Chemical compound OC(=O)C(F)(F)C(F)(F)CF IPFHQCGGKGZQFJ-UHFFFAOYSA-N 0.000 claims description 3
- LZFJEYPNPCYKLY-UHFFFAOYSA-N 2,2,3,3,4-pentafluoropentanoic acid Chemical compound CC(F)C(F)(F)C(F)(F)C(O)=O LZFJEYPNPCYKLY-UHFFFAOYSA-N 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 3
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
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- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
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- 101100369199 Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938) tfe gene Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Peptides Or Proteins (AREA)
Description
(チップカラムのコンディショニング)
メタノール30μL、Buffer SB 30μL、Buffer SA 30μL、Buffer S500-N 30μL、Buffer SA 30μLを表記の順に遠心機を用いて、遠心加速度2,000xgで通液させた。
Buffer U 50μLでサンプルを再溶解し、カラムにロード後1,200xgで通液を行った後、Buffer U 50μL, Buffer SA 50μL, Buffer SB 50μLを2,000xgで通液させ、さらにBuffer SA, Buffer SBそれぞれ50μLを通液させた。上部C18樹脂へのサンプルの吸着と洗浄・脱塩を行い、C18樹脂からのサンプルの溶出後、SCX樹脂へのサンプルの吸着を行っている。
分画後の溶液は、オートサンプラーバイアル(GLScience、TPXスナップバイアル)に分画サンプルを集めた。
集めた分画液をSpeedVacで3時間処理し、乾固させた。20%TFA水溶液 1μLBufferSC 9μLの混合液もしくはBufferSC 10μLでサンプルを再溶解させ、LC-MS用試料溶液とした。この試料溶液についてLC(C18カラム)/MS(Thermo Fisher社 Q Exactive)システムを用いての測定をおこなった。HPLC条件としてC18カラム(ReproSil-Pur C18-AQ, 1.9 μm resin、Dr. Maisch)0.075×300mmに移動相Aとして0.1%蟻酸水、2%アセトニトリル、移動相Bとして90%アセトニトリルを含む0.1%蟻酸水を用いて初期B濃度を5%として、45分間で直線的に30%とし、その後0.1分間で直線的に100%とし、その後移動相Bを100%にして2分間維持させた。もしくは145分間で直線的に30%とし、その後5分間で直線的に100%とし、その後移動相Bを100%にして10分間維持させた。
Claims (11)
- ペプチド又はタンパク質を含む試料を、陽イオン交換基を有する分離媒体に供給する供給工程、前記試料を前記分離媒体に吸着させる吸着工程、酸性イオンペア試薬に濃度勾配を持たせて用いて前記試料を前記分離媒体から溶出させる溶出工程を備えることを特徴とするペプチド又はタンパク質の分画方法。
- 前記イオンペア試薬は揮発性を有することを特徴とする請求項1に記載のペプチド又はタンパク質の分画方法。
- 前記溶出工程において前記イオンペア試薬と塩との混合液を用いることを特徴とする請求項1又は2に記載のペプチド又はタンパク質の分画方法。
- 前記イオンペア試薬が、トリフルオロ酢酸、ペンタフルオロプロピオン酸、ペンタフルオロ酪酸、ペンタフルオロ吉草酸からなる群から選ばれる少なくとも1種であることを特徴とする請求項1から3のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 前記塩が揮発性塩である酢酸アンモニウム、蟻酸アンモニウム、水酸化アンモニウム或いは塩化ナトリウム、塩化カリウムからなる群から選ばれる少なくとも1種であることを特徴とする請求項3に記載のペプチド又はタンパク質の分画方法。
- 前記陽イオン交換基がスルホン酸基、カルボン酸基からなる群から選ばれる少なくとも1種であることを特徴とする請求項1から5のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 前記供給工程において、ペプチド又はタンパク質を含む試料を、逆相官能基を有する逆相分離媒体に通した後に陽イオン交換基を有する分離媒体に供給することを特徴とする請求項1から6のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 前記分離媒体が、陽イオン交換基及び逆相官能基を同時に有することを特徴とする請求項1から6のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 前記分離媒体が、モノリス体であることを特徴とする請求項1から8のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 前記ペプチド又はタンパク質を含む試料が酸化金属により精製されたリン酸化ペプチドであることを特徴とする請求項1から9のうちいずれか1項に記載のペプチド又はタンパク質の分画方法。
- 請求項1から10のうちいずれか1項に記載のペプチド又はタンパク質の分画方法によりペプチド又はタンパク質を含む試料を分画する分画工程、前記分画工程により分画されたペプチド又はタンパク質を含む試料を質量分析装置に供し、前記分画されたペプチド又はタンパク質の質量を測定する工程を含む、ペプチド又はタンパク質の質量分析方法。
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JP2004008027A (ja) * | 2002-06-04 | 2004-01-15 | Japan Science & Technology Corp | cAMPの産生活性を有する新規ペプチド |
GB0306756D0 (en) * | 2003-03-24 | 2003-04-30 | Xzillion Gmbh & Co Kg | Mass labels |
DE10355559A1 (de) * | 2003-11-21 | 2005-06-23 | Orthogen Ag | Transskin |
US7906630B2 (en) * | 2004-04-27 | 2011-03-15 | PerkinElmer Heath Sciences, Inc. | Method for identifying peptides in a biological sample |
JP2010515917A (ja) * | 2007-01-10 | 2010-05-13 | ジーイー・ヘルスケア・バイオ−サイエンシズ・アーベー | クロマトグラフィーレジン |
JP5366234B2 (ja) * | 2007-08-24 | 2013-12-11 | 学校法人関西医科大学 | 扁平上皮がんに対する放射線治療後における予後の予測方法および予後予測用キット |
RU2461564C2 (ru) * | 2008-02-06 | 2012-09-20 | Байокон Лимитид | Способ очистки циклического или нециклического пептида |
JP5214306B2 (ja) * | 2008-04-10 | 2013-06-19 | 学校法人慶應義塾 | リン酸化ペプチド又はリン酸化タンパク質の分離方法 |
MY186089A (en) * | 2009-08-11 | 2021-06-21 | Biocon Ltd | Chromatographic processes and purified compounds thereof |
JPWO2011108451A1 (ja) * | 2010-03-01 | 2013-06-27 | 日本ケミカルリサーチ株式会社 | 遺伝子ノックアウト細胞を用いた組換え体リソソーム酵素の製造方法 |
SG11201602061YA (en) * | 2013-09-17 | 2016-04-28 | Kaneka Corp | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom |
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